Novel human synthetase and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/284,125 which was filed on Apr. 16, 2001 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding a proteinsharing sequence similarity with mammalian synthetases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded protein, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, andcosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

[0003] Synthetases are enzymes that participate in the production ofchemical compounds in the cell. Synthetases have been associated with,inter alia, regulating development, modulating cellular processes, andsignal transduction.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode a novel human protein,and the corresponding amino acid sequence of this protein. The novelhuman synthetase (NHS) described for the first time herein is aadenylsuccinate synthetase that shares structural similarity with animalsynthetases, particularly adenylsuccinate synthetase (REFSEQ accessionnumber: XM058642, which is herein incorporated by reference in itsentirety), the enzyme that catalyzes the first step in the production ofAMP from IMP.

[0005] The novel human nucleic acid (cDNA) sequence described hereinencodes a protein/open reading frame (ORF) of 457 amino acids in length(see SEQ ID NO: 2).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHSs, including small molecules, large molecules, mutant NHSs,or portions thereof, that compete with native NHS, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHSs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHSs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHS sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHS. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHSs. When the unique NHSsequences described in SEQ ID NOS:1-2 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHS sequences described in SEQ IDNOS:1-2 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins which would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses. To these ends,gene trapped knockout ES cells have been generated in murine homologs ofthe described NHSs.

[0007] Additionally, the unique NHS sequences described in SEQ IDNOS:1-2 are useful for the identification of protein coding sequence andmapping a unique gene to a particular chromosome. These sequencesidentify actual, biologically verified, and therefore relevant, exonsplice junctions as opposed to those that may have beenbioinformatically predicted from genomic sequence alone. The sequencesof the present invention are also useful as additional DNA markers forrestriction fragment length polymorphism (RFLP) analysis, and inforensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists, of NHS expression and/or NHS activity that utilize purifiedpreparations of the described NHSs and/or NHS product, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides sequences encoding the describedNHS amino acid sequence.

5. DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHS described for the first time herein is a novel proteinthat can be expressed in human pituitary, lymph node, skeletal muscle,esophagus, pericardium, fetal kidney, fetal lung, tongue, andadenocarcinoma cells.

[0011] The described sequences were compiled from cDNAs prepared andisolated from human liver and placenta mRNAs (Edge Biosystems,Gaithersburg, Md.). The present invention encompasses the nucleotidespresented in the Sequence Listing, host cells expressing suchnucleotides, the expression products of such nucleotides, and: (a)nucleotides that encode mammalian homologs of the described genes,including the specifically described NHS, and the NHS products; (b)nucleotides that encode one or more portions of the NHS that correspondto functional domains, and the polypeptide products specified by suchnucleotide sequences, including, but not limited to, the novel regionsof any active domain(s); (c) isolated nucleotides that encode mutantversions, engineered or naturally occurring, of the described NHS inwhich all or a part of at least one domain is deleted or altered, andthe polypeptide products specified by such nucleotide sequences,including, but not limited to, soluble proteins and peptides in whichall or a portion of the signal sequence is deleted; (d) nucleotides thatencode chimeric fusion proteins containing all or a portion of a codingregion of the NHS, or one of its domains (e.g., a receptor or ligandbinding domain, accessory protein/self-association domain, etc.) fusedto another peptide or polypeptide; or (e) therapeutic or diagnosticderivatives of the described polynucleotides such as oligonucleotides,antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructscomprising a sequence first disclosed in the Sequence Listing.

[0012] As discussed above, the present invention includes: (a) the humanDNA sequences presented in the Sequence Listing (and vectors comprisingthe same) and additionally contemplates any nucleotide sequence encodinga contiguous NHS open reading frame (ORF), or a contiguous exon splicejunction first described in the Sequence Listing, that hybridizes to acomplement of a DNA sequence presented in the Sequence Listing underhighly stringent conditions, e.g., hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., NY, at p. 2.10.3) andencodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of the DNA sequence that encode and express an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encode a functionally equivalent NHS product.Functional equivalents of a NHS include naturally occurring NHSs presentin other species and mutant NHSs whether naturally occurring orengineered (by site directed mutagenesis, gene shuffling, directedevolution as described in, for example, U.S. Pat. No. 5,837,458). Theinvention also includes degenerate nucleic acid variants of thedisclosed NHS polynucleotide sequences.

[0013] Additionally contemplated are polynucleotides encoding a NHS ORF,or its functional equivalent, encoded by a polynucleotide sequence thatis about 99, 95, 90, or about 85 percent similar or identical tocorresponding regions of the nucleotide sequences of the SequenceListing (as measured by BLAST sequence comparison analysis using, forexample, the GCG sequence analysis package using standard defaultsettings).

[0014] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHS gene nucleotide sequences. Such hybridizationconditions may be highly stringent or less highly stringent, asdescribed above. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80, or about 34 toabout 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0015] Alternatively, such NHS oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a micro array or high-throughput“chip” format). Additionally, a series of the described NHSoligonucleotide sequences, or the complements thereof, can be used torepresent all or a portion of the described NHS sequences. Anoligonucleotide or polynucleotide sequence first disclosed in at least aportion of one or more of the sequences of SEQ ID NOS: 1-2 can be usedas a hybridization probe in conjunction with a solid supportmatrix/substrate (resins, beads, membranes, plastics, polymers, metal ormetallized substrates, crystalline or polycrystalline substrates, etc.).Of particular note are spatially addressable arrays (i.e., gene chips,microtiter plates, etc.) of oligonucleotides and polynucleotides, orcorresponding oligopeptides and polypeptides, wherein at least one ofthe biopolymers present on the spatially addressable array comprises anoligonucleotide or polynucleotide sequence first disclosed in at leastone of the sequences of SEQ ID NOS: 1-2, or an amino acid sequenceencoded thereby. Methods for attaching biopolymers to, or synthesizingbiopolymers on, solid support matrices, and conducting binding studiesthereon are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637,5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326,5,424,186, and 4,689,405 the disclosures of which are hereinincorporated by reference in their entirety.

[0016] Addressable arrays comprising at least a portion of the sequencesfirst disclosed in SEQ ID NOS:1-2 can be used to identify andcharacterize the temporal and tissue specific expression of a gene.These addressable arrays incorporate oligonucleotide sequences ofsufficient length to confer the required specificity, yet be within thelimitations of the production technology. The length of these probes iswithin a range of between about 8 to about 2000 nucleotides. Preferablythe probes consist of 60 nucleotides and more preferably 25 nucleotidesfrom the sequences first disclosed in SEQ ID NOS:1-2.

[0017] For example, a series of the described oligonucleotide sequences,or the complements thereof, can be used in chip format to represent allor a portion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

[0018] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprising atleast a portion of the sequences first disclosed in SEQ ID NOS:1-2provides detailed information about transcriptional changes involved ina specific pathway, potentially leading to the identification of novelcomponents or gene functions that manifest themselves as novelphenotypes.

[0019] Probes consisting of at least a portion of the sequences firstdisclosed in SEQ ID NOS:1-2 can also be used in the identification,selection and validation of novel molecular targets for drug discovery.The use of these unique sequences permits the direct confirmation ofdrug targets and recognition of drug dependent changes in geneexpression that are modulated through pathways distinct from the drugsintended target. These unique sequences therefore also have utility indefining and monitoring both drug action and toxicity.

[0020] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-2 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out using atleast a portion of the sequences first disclosed in SEQ ID NOS:1-2 insilico and by comparing previously collected genetic databases and thedisclosed sequences using computer software known to those in the art.

[0021] Thus the sequences first disclosed in SEQ ID NOS:1-2 can be usedto identify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

[0022] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-2. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence.

[0023] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHS gene antisense molecules, useful, for example,in NHS gene regulation and/or as antisense primers in amplificationreactions of NHS gene nucleic acid sequences. With respect to NHS generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences may be used as part of ribozymeand/or triple helix sequences that are also useful for NHS generegulation.

[0024] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0025] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0026] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0027] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An β-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHS.

[0028] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

[0029] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

[0030] Alternatively, suitably labeled NHS nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0031] Further, a NHS homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHS products disclosed herein. The template for thereaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from human or non-human cell lines ortissue known or suspected to express an allele of a NHS gene. The PCRproduct can be subcloned and sequenced to ensure that the amplifiedsequences represent the sequence of the desired NHS gene. The PCRfragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0032] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express a NHS gene, such as, for example, testistissue). A reverse transcription (RT) reaction can be performed on theRNA using an oligonucleotide primer specific for the most 5′ end of theamplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see e.g., Sambrooket al., 1989, supra.

[0033] A cDNA encoding a mutant NHS sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known or suspected to be expressed in an individualputatively carrying a mutant NHS allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal sequence. Using these two primers, the product isthen amplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well-known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHSallele to that of a corresponding normal NHS allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHSgene product can be ascertained.

[0034] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry a mutant NHSallele (e.g., a person manifesting a NHS-associated phenotype such as,for example, obesity, high blood pressure, connective tissue disorders,infertility, etc.), or a cDNA library can be constructed using RNA froma tissue known, or suspected, to express a mutant NHS allele. A normalNHS gene, or any suitable fragment thereof, can then be labeled and usedas a probe to identify the corresponding mutant NHS allele in suchlibraries. Clones containing mutant NHS sequences can then be purifiedand subjected to sequence analysis according to methods well-known tothose skilled in the art.

[0035] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHS allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue can be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against normal NHS product, as described below. For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, NY.

[0036] Additionally, screening can be accomplished by screening withlabeled NHS fusion proteins, such as, for example, alkalinephosphatase-NHS or NHS-alkaline phosphatase fusion proteins. In caseswhere a NHS mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to NHS are likely to cross-react with acorresponding mutant NHS expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well-known in theart.

[0037] The invention also encompasses (a) DNA vectors that contain anyof the foregoing NHS coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHS coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHS coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHS sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0038] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHS, as well as compounds or nucleotide constructs thatinhibit expression of a NHS sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHS (e.g., expression constructs in which NHS coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0039] Alternatively, the gene encoding the NHS can be activated byproviding or overexpressing a transcription factor that directly orindirectly activates the expression of the NHS (see, for example, U.S.application Ser. No. 09/229,007 herein incorporated by reference).

[0040] The NHS or NHS peptides, NHS fusion proteins, NHS nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHSs or inappropriately expressed NHSs for thediagnosis of disease. The NHS proteins or peptides, NHS fusion proteins,NHS nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHS inthe body. The use of engineered host cells and/or animals may offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to the endogenous receptor for an NHS, but can alsoidentify compounds that trigger NHS-mediated activities or pathways.

[0041] Finally, the NHS products can be used as therapeutics. Forexample, soluble derivatives such as NHS peptides/domains correspondingto NHS, NHS fusion protein products (especially NHS-Ig fusion proteins,i.e., fusions of a NHS, or a domain of a NHS, to an IgFc), NHSantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHS-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHS, or a NHS-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the NHS could activateor effectively antagonize the endogenous NHS receptor. Nucleotideconstructs encoding such NHS products can be used to geneticallyengineer host cells to express such products in vivo; these geneticallyengineered cells function as “bioreactors” in the body delivering acontinuous supply of a NHS, a NHS peptide, or a NHS fusion protein tothe body. Nucleotide constructs encoding functional NHS, mutant NHSs, aswell as antisense and ribozyme molecules can also be used in “genetherapy” approaches for the modulation of NHS expression. Thus, theinvention also encompasses pharmaceutical formulations and methods fortreating biological disorders.

[0042] Various aspects of the invention are described in greater detailin the subsections below.

5.1 The NHS Sequences

[0043] The cDNA sequence (SEQ ID NO: 1) and the corresponding deducedamino acid sequence (SEQ ID NO: 2) of the described NHS are presented inthe Sequence Listing. The exons encoding the NHS ORF are apparentlypresent on human chromosome 14 (see GENBANK accession no. AL583722).Accordingly, the described sequences are useful for mapping the codingregions of the human genome.

[0044] The described novel human polynucleotide sequences can be used,among other things, in the molecular mutagenesis/evolution of proteinsthat are at least partially encoded by the described novel sequencesusing, for example, polynucleotide shuffling or related methodologies.Such approaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458which are herein incorporated by reference in their entirety.

[0045] NHS gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate NHS transgenic animals.

[0046] Any technique known in the art may be used to introduce a NHStransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus-mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0047] The present invention provides for transgenic animals that carrythe NHS transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lakso et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

[0048] When it is desired that a NHS transgene be integrated into thechromosomal site of the endogenous NHS gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHSgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHS gene (i.e.,“knockout” animals).

[0049] The transgene can also be selectively introduced into aparticular cell-type, thus inactivating the endogenous NHS gene in onlythat cell-type, by following, for example, the teaching of Gu et al.,1994, Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

[0050] Once transgenic animals have been generated, the expression ofthe recombinant NHS gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHS gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHStransgene product.

[0051] The present invention also provides for “knock-in” animals.Knock-in animals are those in which a polynucleotide sequence (i.e., agene or a cDNA) that the animal does not naturally have in its genome isinserted in such a way that the sequence is expressed. Examples include,but are not limited to, a human gene or cDNA used to replace its murineortholog in the mouse, a murine cDNA used to replace the murine gene inthe mouse, and a human gene or cDNA or murine cDNA that is tagged with areporter construct used to replace the murine ortholog or gene in themouse. Such replacements can occur at the locus of the murine orthologor gene, or at another specific site. Such knock-in animals are usefulfor the in vivo study, testing and validation of, intra alia, human drugtargets, as well as for compounds that are directed at the same andtherapeutic proteins.

5.2 NHS and NHS Polypeptides

[0052] NHS, NHS polypeptides, NHS peptide fragments, mutated, truncated,or deleted forms of NHS, and/or NHS fusion proteins can be prepared fora variety of uses. These uses include, but are not limited to, thegeneration of antibodies, as therapeutics (for treating inflammatory orproliferative disorders, infectious disease, clotting disorders, cancer,etc.) or in the development of therapeutic for treating the same, asreagents in diagnostic assays, the identification of other cellular geneproducts related to a NHS, as reagents in assays for screening forcompounds that can be used as pharmaceutical reagents useful in thetherapeutic treatment of mental, biological, or medical disorders anddiseases. The described NHS is a novel human adenylosuccinatesynthetase.

[0053] The Sequence Listing discloses the amino acid sequence encoded bythe described NHS polynucleotides. The ORF encoding the NHS displays aninitiator methionine in a DNA sequence context consistent with atranslation initiation site.

[0054] The NHS amino acid sequence of the invention includes the aminoacid sequence presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHS homologues from otherspecies are encompassed by the invention. In fact, any NHS encoded bythe NHS nucleotide sequences described above are within the scope of theinvention, as are any novel polynucleotide sequences encoding all or anynovel portion of an amino acid sequence presented in the SequenceListing. The degenerate nature of the genetic code is well-known, and,accordingly, each amino acid presented in the Sequence Listing, isgenerically representative of the well-known nucleic acid “triplet”codon, or in many cases codons, that can encode the amino acid. As such,as contemplated herein, the amino acid sequences presented in theSequence Listing, when taken together with the genetic code (see, forexample, Table 4-1 at page 109 of “Molecular Cell Biology”, 1986, J.Darnell et al. eds., Scientific American Books, New York, N.Y., hereinincorporated by reference) are generically representative of all thevarious permutations and combinations of nucleic acid sequences that canencode such amino acid sequences.

[0055] The invention also encompasses proteins that are functionallyequivalent to the NHS encoded by the presently described nucleotidesequences as judged by any of a number of criteria, including, but notlimited to, the ability to bind and cleave a substrate of a NHS, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHSproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHSnucleotide sequences described above, but which result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions can be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0056] A variety of host-expression vector systems can be used toexpress the NHS nucleotide sequences of the invention. Where, as in thepresent instance, the NHS peptide or polypeptide is thought to be asoluble or secreted molecule, the peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express NHS, or functional equivalent, insitu. Purification or enrichment of NHS from such expression systems canbe accomplished using appropriate detergents and lipid micelles andmethods well-known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of theNHS, but to assess biological activity, e.g., in certain drug screeningassays.

[0057] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHS nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHSnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHS nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHS nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHS nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0058] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHSproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHS, or for raising antibodies to a NHS,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHS coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Pharmacia or American Type Culture Collection) can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

[0059] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign polynucleotidesequences. The virus grows in Spodoptera frugiperda cells. A NHS codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of NHS coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted sequence is expressed (e.g., see Smith etal., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0060] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHS nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing-a NHS product in infected hosts(e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHS nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHS gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHS coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (See Bitter et al.,1987, Methods in Enzymol. 153:516-544).

[0061] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0062] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the NHS sequences described above can be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the NHS product. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds that affectthe endogenous activity of the NHS product.

[0063] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817)genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567;O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colbere-Garapin et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147).

[0064] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺-nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

[0065] Also encompassed by the present invention are fusion proteinsthat direct a NHS to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHSs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching an appropriate signal sequence to a NHSwould also transport a NHS to a desired location within the cell.Alternatively targeting of a NHS or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes: A Practical Approach”, New,R.R.C., ed., Oxford University Press, N.Y., and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures, which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of NHSs to a target site ordesired organ, where they cross the cell membrane and/or the nucleuswhere the NHSs can exert their functional activity. This goal may beachieved by coupling of a NHS to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. Provisional Patent Application Ser. Nos. 60/111,701and 60/056,713, both of which are herein incorporated by reference, forexamples of such transducing sequences), to facilitate passage acrosscellular membranes, and can optionally be engineered to include nuclearlocalization signals.

[0066] Additionally contemplated are oligopeptides that are modeled onan amino acid sequence first described in the Sequence Listing. Such NHSoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHS oligopeptidescan be of any length disclosed within the above ranges and can initiateat any amino acid position represented in the Sequence Listing.

[0067] The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components which naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially isolated orpure protein or polypeptide may be obtained, for example, by extractionfrom a natural source, by expression of a recombinant nucleic acidencoding the protein or polypeptide, or by chemically synthesizing theprotein or polypeptide.

[0068] Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants which accompany it inits natural state. Thus, a polypeptide which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

5.3 Antibodies to NHS Products

[0069] Antibodies that specifically recognize one or more epitopes of aNHS, or epitopes of conserved variants of a NHS, or peptide fragments ofa NHS are also encompassed by the invention. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0070] The antibodies of the invention may be used, for example, in thedetection of NHS in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHS. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHS expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHS-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHS activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

[0071] For the production of antibodies, various host animals may beimmunized by injection with the NHS, an NHS peptide (e.g., onecorresponding to a functional domain of an NHS), truncated NHSpolypeptides (NHS in which one or more domains have been deleted),functional equivalents of the NHS or mutated variant of the NHS. Suchhost animals may include but are not limited to pigs, rabbits, mice,goats, and rats, to name but a few. Various adjuvants may be used toincrease the immunological response, depending on the host species,including, but not limited to, Freund's adjuvant (complete andincomplete), mineral salts such as aluminum hydroxide or aluminumphosphate, chitosan, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, choleratoxin or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0072] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0073] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion Such technologies are described in U.S. Pat. Nos. 5,877,397;6,075,181 and 6,150,584 and their respective disclosures which areherein incorporated by reference in their entirety.

[0074] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHS expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0075] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275,-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0076] Antibodies to a NHS can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHS, using techniqueswell-known to those skilled in the art. (See, e.g., Greenspan & Bona,1993, FASEB J 7(5):437-444; and Nisonoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to a NHS domain andcompetitively inhibit the binding of NHS to its cognate receptor can beused to generate anti-idiotypes that “mimic” the NHS and, therefore,bind and activate or neutralize a receptor. Such anti-idiotypicantibodies or Fab fragments of such anti-idiotypes can be used intherapeutic regimens involving a NHS signaling pathway.

[0077] Additionally given the high degree of relatedness of mammalianNHSs, the presently described knock-out mice (having never seen NHS, andthus never been tolerized to NHS) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHS (i.e., NHS will be immunogenic in NHS knock-outanimals).

[0078] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 2 1 1374 DNA homo sapiens 1 atgtcgggga cccgagcctc caacgaccggccccccggcg caggcggcgt caagcggggg 60 cggctgcagc aggaggcggc ggcgaccggctcccgcgtga cggtggtgct gggcgcgcag 120 tggggggacg agggcaaagg caaggtggtggacctgctgg ccacggacgc cgacatcatc 180 agccgctgcc aggggggcaa caacgccggccacacggtgg tggtggatgg gaaagagtac 240 gacttccacc tgctgcccag cggcatcatcaacaccaagg ccgtgtcctt cattggcaac 300 ggggtggtca tccacttgcc aggcttgtttgaggaagcag agaagaatga aaagaaaggc 360 ctgaaggact gggagaagag gctcatcatctctgacagag cccaccttgt gtttgatttt 420 caccaggctg tcgacggact tcaggaagtgcagcgccagg cacaagaggg gaagaatata 480 ggcaccacca agaagggaat cggaccaacctactcttcca aagctgcccg gacaggcctc 540 cgcatctgcg acctcctgtc agattttgatgagttttcct ccagattcaa gaacctggcc 600 caccagcacc agtcgatgtt ccccaccctggaaatagaca ttgaaggcca actcaaaagg 660 ctcaagggct ttgctgagcg gatcagacccatggtccgag atggtgttta ctttatgtat 720 gaggcactcc acggcccccc caagaagatcctggtggagg gtgccaacgc cgccctcctc 780 gacattgact tcgggaccta cccctttgtgacttcatcca actgcaccgt gggcggtgtg 840 tgcacgggcc tgggcatccc cccgcagaacataggtgacg tgtatggcgt ggtgaaagcc 900 tataccacac gtgtgggcat cggggccttccccaccgagc agatcaacga gattggaggc 960 ctgctgcaga cccgcggcca cgagtggggagtgaccacag gcaggaagag gcgctgcggc 1020 tggctcgacc tgatgattct aagatatgctcacatggtca acggattcac tgcgctggcc 1080 ctgacgaagc tggacatcct ggacgtactgggtgaggtta aagtcggtgt ctcatacaag 1140 ctgaacggga aaaggattcc ctatttcccagctaaccagg agatgcttca gaaggtcgaa 1200 gttgagtatg aaacgctgcc tgggtggaaagcagacacca caggcgccag gaggtgggag 1260 gacctgcccc cacaggccca gaactacatccgctttgtgg agaatcacgt gggagtcgca 1320 gtcaaatggg ttggtgttgg caagtcaagagagtcgatga tccagctgtt ttag 1374 2 457 PRT homo sapiens 2 Met Ser Gly ThrArg Ala Ser Asn Asp Arg Pro Pro Gly Ala Gly Gly 1 5 10 15 Val Lys ArgGly Arg Leu Gln Gln Glu Ala Ala Ala Thr Gly Ser Arg 20 25 30 Val Thr ValVal Leu Gly Ala Gln Trp Gly Asp Glu Gly Lys Gly Lys 35 40 45 Val Val AspLeu Leu Ala Thr Asp Ala Asp Ile Ile Ser Arg Cys Gln 50 55 60 Gly Gly AsnAsn Ala Gly His Thr Val Val Val Asp Gly Lys Glu Tyr 65 70 75 80 Asp PheHis Leu Leu Pro Ser Gly Ile Ile Asn Thr Lys Ala Val Ser 85 90 95 Phe IleGly Asn Gly Val Val Ile His Leu Pro Gly Leu Phe Glu Glu 100 105 110 AlaGlu Lys Asn Glu Lys Lys Gly Leu Lys Asp Trp Glu Lys Arg Leu 115 120 125Ile Ile Ser Asp Arg Ala His Leu Val Phe Asp Phe His Gln Ala Val 130 135140 Asp Gly Leu Gln Glu Val Gln Arg Gln Ala Gln Glu Gly Lys Asn Ile 145150 155 160 Gly Thr Thr Lys Lys Gly Ile Gly Pro Thr Tyr Ser Ser Lys AlaAla 165 170 175 Arg Thr Gly Leu Arg Ile Cys Asp Leu Leu Ser Asp Phe AspGlu Phe 180 185 190 Ser Ser Arg Phe Lys Asn Leu Ala His Gln His Gln SerMet Phe Pro 195 200 205 Thr Leu Glu Ile Asp Ile Glu Gly Gln Leu Lys ArgLeu Lys Gly Phe 210 215 220 Ala Glu Arg Ile Arg Pro Met Val Arg Asp GlyVal Tyr Phe Met Tyr 225 230 235 240 Glu Ala Leu His Gly Pro Pro Lys LysIle Leu Val Glu Gly Ala Asn 245 250 255 Ala Ala Leu Leu Asp Ile Asp PheGly Thr Tyr Pro Phe Val Thr Ser 260 265 270 Ser Asn Cys Thr Val Gly GlyVal Cys Thr Gly Leu Gly Ile Pro Pro 275 280 285 Gln Asn Ile Gly Asp ValTyr Gly Val Val Lys Ala Tyr Thr Thr Arg 290 295 300 Val Gly Ile Gly AlaPhe Pro Thr Glu Gln Ile Asn Glu Ile Gly Gly 305 310 315 320 Leu Leu GlnThr Arg Gly His Glu Trp Gly Val Thr Thr Gly Arg Lys 325 330 335 Arg ArgCys Gly Trp Leu Asp Leu Met Ile Leu Arg Tyr Ala His Met 340 345 350 ValAsn Gly Phe Thr Ala Leu Ala Leu Thr Lys Leu Asp Ile Leu Asp 355 360 365Val Leu Gly Glu Val Lys Val Gly Val Ser Tyr Lys Leu Asn Gly Lys 370 375380 Arg Ile Pro Tyr Phe Pro Ala Asn Gln Glu Met Leu Gln Lys Val Glu 385390 395 400 Val Glu Tyr Glu Thr Leu Pro Gly Trp Lys Ala Asp Thr Thr GlyAla 405 410 415 Arg Arg Trp Glu Asp Leu Pro Pro Gln Ala Gln Asn Tyr IleArg Phe 420 425 430 Val Glu Asn His Val Gly Val Ala Val Lys Trp Val GlyVal Gly Lys 435 440 445 Ser Arg Glu Ser Met Ile Gln Leu Phe 450 455

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence that: (a) encodes the amino acid sequence shown inSEQ ID NO: 2; and (b) hybridizes under stringent conditions to thenucleotide sequence of SEQ ID NO: 1 or the complement thereof.
 2. Anisolated nucleic acid molecule according to claim 1 wherein saidnucleotide sequence is present in cDNA.
 3. An isolated nucleic acidmolecule encoding the amino acid sequence described in SEQ ID NO:2.